CN103283604B - Method for removing endophytic bacteria of explants in lily tissue culture - Google Patents
Method for removing endophytic bacteria of explants in lily tissue culture Download PDFInfo
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- CN103283604B CN103283604B CN201310225467.1A CN201310225467A CN103283604B CN 103283604 B CN103283604 B CN 103283604B CN 201310225467 A CN201310225467 A CN 201310225467A CN 103283604 B CN103283604 B CN 103283604B
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Abstract
The invention provides a method for removing the endophytic bacteria of explants in lily tissue culture. According to the method, pre-treatment, endophytic bacterium leaching, surface disinfection, inoculated culture and continuous disinfection are performed, and the lily explants are soaked by sterile deionized water, so that the superficial cells of the explants can be broken, and the endophytic bacteria overflow and are easy to eliminate; and continuous surface disinfection is adopted, and the contamination rate of the endophytic bacteria of the lily explants can be controlled to be about 5% after three times of disinfection, so that the problem of serious contamination of the endophytic bacteria is solved for the industry of lily tissue culture. Due to adoption of the method for removing the endophytic bacteria of the explants in lily tissue culture, the endophytic bacteria in lily flakes can be effectively killed; a preparation method easy to operate and good in effect, for the explants, is provided for lily tissue culture; and moreover, materials and cost are greatly saved and reduced to a large extent in the whole process, and the endophytic bacteria in the lily explants can be furthest removed. Compared with the conventional method, the method provided by the invention has the advantages that the lily explants are greatly saved, and the investment of manpower and material resources is reduced.
Description
Technical field
The present invention relates to during a kind of Lilium Tissue is cultivated the method removing explant endophyte.
Background technology
Lily is Liliaceae lilium perennial napiform root class flowers, is one of current high-grade cut-flower most popular in the world.Except ornamental value, lily also has abundant nutritive value and important medical value.Its Sterile culture mode is for point planting clove, and some kinds can use scale cuttage, but year reproduction rate low, limited amount, and lily susceptible viral infects.The kind ball quality of domestic natural propagation is general not good, can not year after year using with detoxification kind ball of import, causes the domestic and international market vacancy of lily very large, constantly sells well at Market of Fresh Cut Flower.Appliable plant tissue culture and rapid propagation method to some extent solves the phenomenon of lily high-quality bulb shortage, and the quick propagating technology about lily is domestic has report more.
But the initial stage of cultivating at Lilium Tissue has a common problem, that be exactly in lily explant endophyte cannot effectively remove, particularly lily bud scale.At present, Lilium Tissue mainly uses alcohol, mercuric chloride, hypochlorous acid etc. to carry out the surface sterilization of lily explant in cultivating, constantly observe in initial-stage culture, every removal immediately having pollution, often can only remain little not by endophytic bacterial contamination explant, so not only waste material but also waste manpower and materials.
The problems referred to above should be paid attention in Lilium Tissue incubation and the problem solved.
Summary of the invention
The object of this invention is to provide during a kind of Lilium Tissue is cultivated the method removing explant endophyte and solve the initial stage of cultivating at Lilium Tissue existed in prior art, in lily explant, particularly in lily bud scale, the problem that endophyte cannot effectively be removed.
Technical solution of the present invention is:
Lilium Tissue removes a method for explant endophyte in cultivating, comprise the following steps:
A, pretreatment: lily bulb clean water is removed surperficial silt, peel scale and again clean, in super-clean bench, lily bud scale is cut into 0.5 ~ 6.0cm
2fritter;
B, endophyte leach: gained lily bud scale fritter in step a is put into the container that aseptic deionized water is housed, and it is place 0.5 ~ 8h in 25 ~ 37 DEG C of environment that airtight container is placed on temperature, carries out endophyte leaching.
C, surface sterilization: the lily bud scale fritter soaked by aseptic deionized water in step b in super-clean bench carries out surface sterilization, the concrete steps of surface sterilization are: taken out from aseptic deionized water by lily bud scale fritter in super-clean bench, after sterile water wash 1 ~ 3 time, the ethanol putting into 75% soaks 0.1 ~ 0.5min, again by after sterile water wash 2 ~ 3 times, after putting into the mercuric chloride solution 1 ~ 30min of concentration 0.1%, then by sterile water wash 5 ~ 8 times; After surface sterilization, excision lily bud scale fritter epidermis.
D, inoculated and cultured: be inoculated in solid culture medium by the lily bud scale fritter after surface sterilization in step c, cultivate and observe with or without endophyte; The formula of the solid culture medium that inoculation uses is: MS+0.5 ~ 2.0mg/L 6-BA+0.01 ~ 0.5mg/L NAA+0.1 ~ 0.5mg/L carbendazim+25 ~ 35g/L sucrose+5 ~ 10g/L agar powder PH5.0 ~ 6.8; The culture environment using solid culture medium is temperature 25 ~ 27 DEG C, and periodicity of illumination is 10 ~ 14h/d;
E, continuous sterilization:
Observation is cultivated after the inoculation of steps d, if grow lily endophyte in medium, take out lily bud scale and carry out the follow-up continued access kind observation of continuous sterilization, repeat that continuous sterilization is carried out to the lily bud scale growing lily endophyte and observe, until grow without lily endophyte in medium with inoculation;
Continuous sterilization is specially: taken out from solid culture by lily bud scale in super-clean bench, after sterile water wash 2 ~ 3 times, the ethanol putting into 75% soaks 0.1 ~ 0.3min, sterile water wash 2 ~ 3 times, put into the mercuric chloride solution 1 ~ 15min of 0.1% again, sterile water wash 4 ~ 7 times.
Preferably, in the pretreatment of described step a, lily bud scale is removed epidermis, and cut into 2.0 ~ 4.0 cm
2fritter.
The invention has the beneficial effects as follows: a kind of Lilium Tissue of the present invention removes the method for explant endophyte in cultivating, effectively can kill the endophyte in lily bud scale, the tissue cultures for lily provides a kind of easy to operate, effective explant preparation method.This kind of Lilium Tissue removes the method for explant endophyte in cultivating, adopt aseptic deionized water to soak lily explant, explant cells of superficial layer can be made to break, endophyte overflows, and is easy to remove.And adopting continuous surface to sterilize, the endophytic bacterial contamination rate of lily explant after three times of sterilizing can control about 5%, cultivates industry solve the serious problem of endophytic bacterial contamination for Lilium Tissue.This kind of Lilium Tissue removes the method for explant endophyte in cultivating, simple to operate, and use instrument or reagent to be staple in Plant Tissue Breeding, person skilled all can easily use the method.This kind of Lilium Tissue removes the method for explant endophyte in cultivating, in whole process, save material largely, reduce costs, and the endophyte that can remove to greatest extent in lily explant, save lily explant greatly compared with the method for routine, decrease the input of manpower and materials.
Embodiment
The following detailed description of the preferred embodiments of the present invention.
Lilium Tissue removes a method for explant endophyte in cultivating, comprise the following steps:
A, pretreatment: lily bulb clean water is removed surperficial silt, peel scale and again clean, be cut into small pieces by lily bud scale in super-clean bench;
B, endophyte leach: gained lily bud scale fritter in step a is put into the container that aseptic deionized water is housed, places and leached by endophyte after seal of vessel;
C, surface sterilization: the lily bud scale fritter soaked by aseptic deionized water in step b in super-clean bench carries out surface sterilization;
D, inoculated and cultured: be inoculated in solid culture medium by the lily bud scale fritter after surface sterilization in step c, cultivate and observe with or without endophyte;
E, continuous sterilization: after the inoculation of steps d, cultivate observation, if grow lily endophyte in medium, take out lily bud scale and carry out the follow-up continued access kind observation of continuous sterilization, repeat to carry out continuous sterilization to the lily bud scale growing lily endophyte to observe, until grow without lily endophyte in medium with inoculation.
The present embodiment removes the method for explant endophyte in providing a kind of Lilium Tissue to cultivate, by pretreatment, endophyte leaching, surface sterilization, inoculated and cultured, continuous sterilization, realize the effective disinfection to lily bulb, endophytic bacterial contamination rate is controlled in less scope.
This kind of Lilium Tissue removes the method for explant endophyte in cultivating, first, lily bulb clean water is removed surperficial silt, peel scale and again clean, in super-clean bench, lily bud scale is cut into 0.5 ~ 6.0cm
2, be preferably 2.0 ~ 4.0 cm
2fritter, completes the pretreatment to lily bulb; Gained lily bud scale fritter is put into the container that aseptic deionized water is housed, and is place 4h in 24-28 DEG C of environment to be leached by endophyte in temperature after seal of vessel; In super-clean bench, lily bud scale fritter is taken out from aseptic deionized water, after sterile water wash 1 ~ 3 time, the ethanol putting into 75% soaks 0.1 ~ 0.5min, again by after sterile water wash 2 ~ 3 times, put into the mercuric chloride solution 1 ~ 30min of concentration 0.1%, preferably after 8 ~ 15min, then by sterile water wash 5 ~ 8 times, after surface sterilization, excision lily bud scale fritter epidermis; Lily bud scale fritter after surface sterilization is inoculated in solid culture medium, under temperature 25 ~ 27 DEG C, periodicity of illumination 12h/d condition, cultivates observation occur with or without endophyte; After the inoculation of lily bud scale fritter, cultivate observation, if grow lily endophyte in medium, take out scale immediately, in super-clean bench by lily bud scale with after sterile water wash 2 times, the ethanol putting into 75% soaks 15s, sterile water wash 2 times, then the mercuric chloride solution 8min putting into 0.1%, sterile water wash 6 times, after having sterilized, continue to cultivate and observe, if again there is endophyte, repeat continuous sterilization, and so forth until without lily endophytic bacterial contamination in medium.
Statistical data by experiment verifies that the beneficial effect of the present embodiment is as follows further:
The present embodiment method carries out the experiment removing endophyte in scale of one-time surface sterilization, secondary surface sterilization to oriental hybrid lily, rope nation lily, lanzhou lily, and compares with remove endophyte and the not treated endophyte in scale that removes in scale after existing method process.
In existing method, explant sterilization is: 75% alcohol-pickled 0.2 ~ 0.5min, and sterile water wash 2 ~ 3 times, 0.1% mercuric chloride soaks 10 ~ 20min, sterile water wash 5 ~ 7 times.Do not process, the Lilium Tissue of existing method, the present embodiment method cultivate in the percentile experimental result of endophytic bacterial contamination as shown in table 1.From the pollution percentage statistical data of endophyte in table 1, the pollution percentage of the endophyte of the oriental hybrid lily after the process of the present embodiment method, rope nation lily, lanzhou lily very significantly lower than existing method process with do not process, effectively the endophytic bacterial contamination rate of lily explant is controlled about 5%, cultivate industry for Lilium Tissue and solve the serious problem of endophytic bacterial contamination.
The percentile experimental result of endophytic bacterial contamination in the cultivation of table 1 Lilium Tissue
Claims (2)
1. Lilium Tissue removes a method for explant endophyte in cultivating, and it is characterized in that, comprises the following steps:
A, pretreatment: lily bulb clean water is removed surperficial silt, peel scale and again clean, in super-clean bench, lily bud scale is cut into 0.5 ~ 6.0cm
2fritter;
B, endophyte leach: gained lily bud scale fritter in step a is put into the container that aseptic deionized water is housed, and it is place 0.5 ~ 8h in 25 ~ 37 DEG C of environment that airtight container is placed on temperature, carries out endophyte leaching;
C, surface sterilization: the lily bud scale fritter soaked by aseptic deionized water in step b in super-clean bench carries out surface sterilization, the concrete steps of surface sterilization are: taken out from aseptic deionized water by lily bud scale fritter in super-clean bench, after sterile water wash 1 ~ 3 time, the ethanol putting into 75% soaks 0.1 ~ 0.5min, again by after sterile water wash 2 ~ 3 times, after putting into the mercuric chloride solution 1 ~ 30min of concentration 0.1%, then by sterile water wash 5 ~ 8 times; After surface sterilization, excision lily bud scale fritter epidermis;
D, inoculated and cultured: be inoculated in solid culture medium by the lily bud scale fritter after surface sterilization in step c, cultivate and observe with or without endophyte; The formula of the solid culture medium that inoculation uses is: MS+0.5 ~ 2.0mg/L 6-BA+0.01 ~ 0.5mg/L NAA+0.1 ~ 0.5mg/L carbendazim+25 ~ 35g/L sucrose+5 ~ 10g/L agar powder PH5.0 ~ 6.8; The culture environment using solid culture medium is temperature 25 ~ 27 DEG C, and periodicity of illumination is 10 ~ 14h/d;
E, continuous sterilization:
Observation is cultivated after the inoculation of steps d, if grow lily endophyte in medium, take out lily bud scale and carry out the follow-up continued access kind observation of continuous sterilization, repeat that continuous sterilization is carried out to the lily bud scale growing lily endophyte and observe, until grow without lily endophyte in medium with inoculation;
Continuous sterilization is specially: taken out from solid culture by lily bud scale in super-clean bench, after sterile water wash 2 ~ 3 times, the ethanol putting into 75% soaks 0.1 ~ 0.3min, sterile water wash 2 ~ 3 times, put into the mercuric chloride solution 1 ~ 15min of 0.1% again, sterile water wash 4 ~ 7 times.
2. Lilium Tissue as claimed in claim 1 removes the method for explant endophyte in cultivating, and it is characterized in that: in the pretreatment of described step a, lily bud scale is removed epidermis, and cuts into 2.0 ~ 4.0 cm
2fritter.
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| CN107022510A (en) * | 2017-06-06 | 2017-08-08 | 昆明学院 | A kind of separation method of endophyte of plant |
| CN107258539B (en) * | 2017-07-06 | 2019-06-07 | 四川省中医药科学院 | A method of control taipei fritillary bulb tissue cultures endophytic bacterial contamination |
| CN112868787A (en) * | 2021-01-29 | 2021-06-01 | 陕西科技大学 | Plant-based probiotic yogurt and preparation method thereof |
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| KR20040013424A (en) * | 2002-08-06 | 2004-02-14 | 주식회사 네오바이오 | Method for mass production of lily |
| CN100333634C (en) * | 2005-06-17 | 2007-08-29 | 云南格桑花卉有限责任公司 | Method for cultivating oriental lily test tube bulb |
| CN100363058C (en) * | 2006-03-21 | 2008-01-23 | 浙江理工大学 | Sterilizing method for separating plant endogenous fungus |
| JP2007259749A (en) * | 2006-03-28 | 2007-10-11 | Kirin Agribio Co Ltd | Method for proliferating plant, characterized by ethylene treatment |
| CN101658135B (en) * | 2009-09-02 | 2011-08-03 | 北京林业大学 | Method for desorbing lily bulb virus |
| CN102440185B (en) * | 2010-10-14 | 2015-05-27 | 赵祥云 | Fast culturing method for lily virus-free seed ball |
| CN102870676B (en) * | 2012-09-18 | 2014-01-08 | 龙山县生物科技开发公司 | Longshan lily high-efficient detoxification process |
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Inventor after: Zhang Benhou Inventor after: Hu Wenjie Inventor after: Chen Jishuang Inventor after: Hu Yanhua Inventor after: Jia Mingliang Inventor before: Zhang Benhou Inventor before: Chen Jishuang Inventor before: Hu Yanhua Inventor before: Jia Mingliang |
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